Process for production of a regioselective membrane

ABSTRACT

A process for production of a microporous affinity membrane having regioselective affinity for compounds in blood or other biologically active fluids to be removed during purification of blood or said fluids is disclosed, as well as a microporous affinity membrane produced by said process, an adsorption device containing such a microporous affinity membrane, and use of such a microporous affinity membrane.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a process for production of amicroporous affinity membrane having regioselective affinity forcompounds in blood or other biologically active fluids to be removedduring purification of blood or said fluids, to a microporous affinitymembrane produced by said process, to an adsorption device containingsuch a microporous affinity membrane, and to use of such a microporousaffinity membrane.

BACKGROUND ART

Microporous hollow fibre membranes and flat sheet membranes are examplesof microporous affinity membranes having a blood side and a filtrateside. Such membranes are well known for analytical, diagnostic ortherapeutical purposes. For example, such microporous hollow fibremembranes and flat sheet membranes are useful for the treatment of bloodor other biologically active fluids with a view to eliminating undesiredcompounds therefrom, i.e. in therapeutic apheresis. Microporous hollowfibre membranes are normally composed of a bundle of separatemicroporous hollow fibres. For detoxification of whole blood, e.g.dialysis and plasmapheresis, the membrane bundle is normally potted ateach end of a polycarbonate tube fitted with two ports in a shell. Theblood is normally extracorporeally pumped through a lumen representingthe blood side, of each fibre, and a part of the blood plasmapenetrates, i.e. is filtrated, through the pores of the fibre wall intoan outer compartment representing the filtrate side, surrounding eachfibre in the bundle. The concentrated blood containing blood cells, toolarge to enter the pores, and the remaining non-filtered part of bloodplasma passes through the lumen. In a venous blood line the filtratedblood plasma stream is normally added to the non-filtered blood streamand returned to the patient.

With a view to eliminating undesired compounds from the blood, thesurfaces and pores of the microporous affinity membranes, e.g.microporous hollow fibre membranes and flat sheet membranes, areprovided with activated sites or ligands specific for binding to theundesired blood compounds to be eliminated. Such activated sites orligands are normally based on or bound to functional groups, e.g. amino,carboxy, or sulfonic acid groups, on the microporous membrane surface.The undesired compounds to be eliminated from the blood are normallytoxins of different kinds, e.g. bacterial derived toxins. Furtherexamples of such undesired compounds are presented below.

The lumen surfaces on the blood side of microporous hollow fibremembranes, the surfaces on the blood side of flat sheet membranes, thesurfaces of the pores and the surfaces on the filtrate side of suchmembranes are often provided with such activated sites or ligands,particularly for purification of blood or biologically active fluids.

In blood purification applications activated sites or ligands, e.g.positive amino groups as functional groups for heparin or endotoxinadsorption, on the surface on the blood side of such membranes mayactivate certain blood constituents, e.g. thrombocytes. In such a case,these blood constituents are activated and/or adhered to the ligands andare significantly reduced from the blood. Such an adhesion is undesired.Other blood constituents, e.g leucocytes, red blood cells and proteins,may in some extent also be adhered to such ligands or activated sites onthe blood side of the membrane.

This undesired activation of blood constituents in such membranes hassince long been a great problem, in particular the accompanyingundesired elimination of thrombocytes from the blood. Several attemptshave been made to solve this problem to prepare microporous hollow fibremembranes and flat sheet membranes lacking the above-mentioned ligandsor activated sites on the blood side of the membrane, but so far onlycomplicated processes requiring large amounts of reaction chemicals andsolvents have been found. Moreover, these processes are also expensive,ineffective and not environmental friendly, thereby creating problemshighly needed to solve.

WO 80/02805 describes, inter alia, a process for the treatment of and/orremoval of undesired compounds from whole blood and a membrane therefor.A biologically activated material is immobilised, i.e. ligands arearranged in the pores, and/or on the surface of said membrane that facesaway from said whole blood, i.e. faces the filtrate side of themembrane. Further, processes for immobilising different kinds ofbiologically active material, i.e. ligands, by treatment with chemicalsare disclosed. Thus, an asymmetric immobilisation, i.e. creation ofregioselective affinity, is disclosed with a view to avoiding contactbetween blood corpuscles and the immobilising reagent and, thus, pyrogenand/or anaphylactic reactions.

U.S. Pat. No. 5,868,936, WO 97/48483, U.S. Pat. No. 5,766,908, andEP-A2-0,341,413 disclose immobilising techniques for attaching ligandsto the surface of the pores in hollow fibre membranes.

U.S. Pat. No. 6,090,292 discloses an asymmetric dialysis hollow fibrecoated with albumin essentially on the side facing away from the blood,i.e. facing the filtrate side.

Plasma treatment is known as an effective method for modification ofsurfaces. It is, inter alia, used to increase the wettability and thusthe adsorption properties of surfaces.

EP-A1-0,683,197, U.S. Pat. No. 6,022,902, U.S. Pat. No. 5,591,140, andU.S. Pat. No. 6,013,789 disclose treatment of a surface with plasma witha view to immobilising certain ligands.

U.S. Pat. No. 5,597,456 discloses atmospheric pressure plasma treatmentof surfaces of medical devices.

EP-A2-0,695,622 discloses plasma modification of flat porous articlesusing low pressure plasma treatment.

SUMMARY OF THE INVENTION

The object of the present invention is to solve the above problem withdefective procedures for production of microporous affinity membraneshaving regioselective affinity for undesired compounds in blood or otherbiologically active fluids with a view to avoiding undesired activationof constituents in blood or other biologically active fluids inmicroporous affinity membranes during the purification treatment ofblood or said fluids.

This object is achieved with a microporous affinity membrane, producedby a process for production of a microporous affinity membrane havingregioselective affinity for compounds in blood or other biologicallyactive fluids to be removed during purification of blood or said fluids,wherein a microporous affinity membrane substrate having a blood sideand a filtrate side is subjected to one or more cycles of plasmaignition in the presence of a gas mixture comprising a functional groupcontaining modifying gas, wherein functional groups are regioselectivelybound to pore surfaces of the microporous affinity membrane substrate.In a further process step ligands having affinity for said compounds inblood or said fluids may be bound to the functional groups.

In one embodiment functional groups are also regioselectively bound tosurfaces on the filtrate side of the microporous affinity membranesubstrate.

The present invention also relates to a microporous affinity membraneproduced by said process, to an adsorption device containing such amicroporous affinity membrane and to use of such a microporous affinitymembrane.

Other objects, features, advantages and preferred embodiments of thepresent invention will become more apparent from the following detaileddescription when taken in conjunction with the drawings and the appendedclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a system comprising a microporous membrane adsorptiondevice with regioselectively functionalized microporous hollow fibremembranes produced according to a preferred embodiment of the presentinvention and having functional groups with ligands bound thereto.Further, in the right part of FIG. 1 such a membrane is shown in anenlarged cross-sectional view.

FIG. 2 a shows outside plasma treatment of a membrane substrate at lowpressure.

FIG. 2 b shows outside plasma treatment of a membrane substrate at highpressure.

FIG. 2 c shows inside plasma treatment of a membrane substrate at lowpressure.

FIG. 2 d shows inside plasma treatment of a membrane substrate at highpressure.

FIG. 3 shows plasma treatment of a microporous flat sheet membranesubstrate according to another embodiment of the present invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In a preferred embodiment the present invention relates to a process forproduction of a microporous hollow fibre membrane having regioselectiveaffinity.

In another preferred embodiment the present invention relates to aprocess for the preparation of a microporous flat sheet membrane havingregioselective affinity.

Throughout the application text and the claims the followingabreviations are used.

-   -   PES=polyethersulfone    -   PVP=polyvinylpyrrolidone    -   PP=polypropylene    -   DACH=diaminocyclohexane    -   DETA=diethylenetriamine    -   ESCA=electrospectroscopy for chemical analysis    -   PFBA=pentafluorobenzaldehyde    -   sccm=standard cubic centimeter

The term “functional group containing modifying gas” or “modifying gas”used throughout the application text means the gaseous form of themolecule leading to surface modification during gas plasma treatment. Inthe gas plasma these molecules comprising the functional groups areconverted to activated species, i.e. radicals or ions. During the gasplasma treatment there is a surface retention of functional groupsresulting in a functional membrane surface, i.e. a membrane withregioselective affinity, having the ability to covalently bind differentligands.

The term “blood” used throughout the application text is intended tocover whole blood and different modifications thereof in which one ormore of the constituents thereof have been separated off.

The term “other biologically active fluids” used throughout theapplication text means pharmaceutically useful solutions orpharmaceutical preparations which contain a biologically activecomponent, such as a coagulation factor.

The term “blood side” used throughout the application text means themembrane side on which blood or another biologically active fluid isbrought to flow during purifycation by use of a microporous affinitymembrane, i.e. either the outer (shell) side or the inner (lumen) sideof a microporous hollow fibre membrane, and any of the both sides of amicroporous flat sheet membrane.

The term “filtrate side” used throughout the application text means themembrane side on which the filtered part of blood or anotherbiologically active fluid reaches after having passed through the poresof a microporous affinity membrane, i.e. either the outer (shell) sideor the inner (lumen) side of a microporous hollow fibre membrane, andany of the both sides of a microporous flat sheet membrane.

The term “compound in blood . . . ” used throughout the application textmeans an undesired compound intended to be removed from the blood.

The terms “blood constituent” and “constituents in blood” usedthroughout the application means components normally existing in blood,e.g. different blood cells and proteins.

The term “gas mixture” used throughout the application text means themixture between modifying gas and carrier gas, but is also used, forsimplicity reasons, for the embodiment when the carrier gas is absent.

The term “gas plasma mixture” used throughout the application means themedium resulting from the plasma ignition of the gas mixture andcontaining the activated species providing the binding of functionalgroups to the surfaces in question.

The terms “microporous affinity membrane substrate” and “membranesubstrate” used throughout the application text means an untreated, notfunctionalised microporous affinity membrane, i.e. lackingregioselective affinity and intended as a start material in the processaccording to the present invention.

The term “microporous hollow fibre membrane” used throughout theapplication text is intended to cover everything from one microporoussingle hollow fibre up to several single hollow fibres and one or morebundles of such microporous hollow fibres, each fibre having a filtrateside and a blood side.

The term “microporous flat sheet membrane” used throughout theapplication text means a micropore containing flat membrane having afiltrate side and a blood side.

In one preferred embodiment of the present invention microporous hollowfibre membranes are regioselectively modified or functionalised only onthe outer surface, i.e. the filtrate side, and on the surfaces withinthe pores in an improved way compared to known techniques.

The membrane lumen surface, i.e. on the blood side, which comes intocontact with whole blood when the membranes are used for blood treatmentin therapeutic apheresis, is to remain unmodified. This is achieved byavoiding affinity on the blood side, thereby inhibiting the interactionbetween certain blood constituents and the ligands bound to functionalgroups introduced regioselectively during the membrane modificationprocess. This is an important requirement for selective removal ofcompounds from whole blood or other biologically active fluids within amembrane adsorption device.

Referring to FIG. 1 the right part thereof shows a preferred embodimentof a regioselective microporous hollow fibre membrane for treatment ofblood in an enlarged cross-sectional view. The flow of blood is markedwith an arrow on the blood side. The membrane wall pores connects theblood side with the filtrate side. The flow of blood plasma containingthe compounds to remove from blood is marked with arrows in the pores.On the surfaces of the pores and on the outer surface on the filtrateside functional groups, to which ligands are attached, have been bound.To some of said ligands compounds to be eliminated have been bound. Asappears, no functional groups are attached to the lumen surface on theblood side.

A major advantage of the present invention compared to prior art, e.g.WO 80/02805, is that the need for reaction chemicals and solvents ishighly reduced and that the total costs, e.g. the cost for disposal ofchemicals, is lowered. Moreover, the present invention provides a moreenvironmental friendly process compared to prior art processes for theproduction of such regioselective membranes. The present invention doesnot require any organic solvent or chemicals that needs to be eliminatedafter the treatment, i.e. the gas mixture used reacts totally and noside products are left to be taken care of afterwards.

Further advantages of the present invention include that the microporousaffinity membranes having regioselective affinity are much easier tomanufacture compared to the conventional wet-chemical approaches. Thisis due to the gas plasma treatment process. Moreover, the presentinvention provides high versatility in that a variety of differentfunctional groups can be arranged to immobilise compounds to beeliminated. This is possible due to independence of the chemicals usedin prior art processes. By means of the gas plasma treatment it ispossible to introduce reactivity in almost all molecules as long as themolecules can ignite to plasma, why a wide variety of functional groupsmay be chosen. Further, high efficiency due to improved mass transportproperties is obtained, i.e. convective transport of blood compounds toeliminate, e.g. toxins, to the binding sites, i.e. ligands, compared tothe corresponding diffusion transport in affinity columns.

In another preferred embodiment of the present invention a microporousflat sheet membrane having regioselective affinity is produced with aprocess corresponding to the process for preparing microporous hollowfibre membranes having corresponding properties. This process isdescribed in detail below, e.g. in Example 3.

The functional groups to be introduced on the membrane substratesurfaces of interest are preferably amino groups originating from suchmolecules as amino compounds (diamines, triamines), e.g.diaminocyclohexane (DACH) and diethylenetriamine (DETA), preferablydiaminocyclohexane, but also from all organic precursors with primaryamino groups or mixtures of hydrogen with nitrogen or ammonia, providedtheir vapour pressure is high enough to give a substantial amount of themolecule containing the functional groups in the vapour phase. Further,other functional groups than amino groups can be introduced, e.g.carboxyl, hydroxyl, sulfonic acid, ester or epoxy groups, whenprecursors comprising corresponding functions are used instead ofcompounds containing amino functions.

The microporous affinity membranes produced according to the presentinvention are made of a biocompatible polymeric material, e.g.polyethersulfone (PES), polyvinylpyrrolidone (PVP), polypropylene (PP),polysulfone (PSU), polymethylmethacrylate (PMMA), polycarbonate (PC),polyacrylonitrile (PAN), polyamide (PA), polytetrafluorethylene (PTFE),cellulose acetate (CA), cellulose nitrate or regenerated cellulose.

The inner diameter of the hollow fibres is normally 200-1000 μm, thewall thickness is normally 20-200 μm and the pore diameter 0.1-2.0 μm.The fibres are normally arranged in modules e.g. containing a bundle of10 to more than 1000 fibres, but single hollow fibres are also possibleto treat. Experimental modules contain 10-100 fibres. Final modules forblood treatment contain more than 1000 fibres. Modules with more fibresmay also be modified according to this procedure.

According to the present invention the hollow fibres used for themicroporous hollow fibre membrane are preferably made of a blend ofpolyethersulfone and polyvinylpyrrolidone with an inner diameter of 330μm, a wall thickness of 110 μm and a pore diameter of 0.4 μm.

The flat sheet membrane is preferably made of a mixture ofpolyethersulfone and polyvinylpyrrolidone with a wall thickness of20-200 μm, preferably 110 μm, and a pore diameter of 0.1-0.8 μm,preferably 0.4 μm.

As stated above, the regioselective introduction of amino groups, i.e.the preferred functional groups, only on the pore surfaces, in practicegradually less towards the blood side, and on the filtrate side, but notat all on the blood side of the microporous affinity membrane substrate,is achieved by gas plasma treatment of the membrane substrate,preferably using DACH or DETA, most preferably DACH, as the functionalgroup containing modifying gas, and a stabilising carrier gas, which ischemically inert during the gas plasma reaction. Preferably helium isused as carrier gas due to the wide pressure range used for ignition ofgas plasma. The use of low gas plasma power is beneficial with respectto the preservation of the functional groups. As alternative carriergases nitrogen, hydrogen and argon or corresponding mixtures may beused. A further possibility is to work without any carrier gas. Duringthe gas plasma treatment this mixture of modifying gas and carrier gasincludes the activated species described above and provides theregioselective introduction of the amino groups on the surfaces ofinterest, however, not on the blood side of the microporous affinitymembrane substrate, due to deactivation of activated species on the wayfrom the plasma glow discharge zone to the blood side. The proportionbetween the functional group containing modifying gas and the carriergas is normally 1:10-1:1, preferably 1:4.

The most important parameters are the direction of the gas plasmamixture flow in relation to the membrane substrate to be treated, themean free path length of the activated species and the flow rate of thegas plasma mixture.

The ligands to be bound to the functional groups introduced on thesurface of the membrane substrate filtrate side and on pore surfaces arechosen dependent on the type of compounds to be removed from the bloodor any other biologically active fluid. Examples of ligands areproteins, peptides, amino acids, carboxylic acids, oligonucleotides andmixtures of two or more thereof or any other convenient biomolecules.The ligands are added to the functional groups in a separatewet-chemical process, known per se.

Some basic principles behind the plasma ignition (plasma glow discharge)processes used in connection with the present invention will now bediscussed.

Plasma can be ignited when the dimension of the gas containment is muchhigher than the mean free path length at a given gas pressure. The meanfree path length is inversely proportional to the gas pressure. In thelow pressure case the mean free path length is dependent on the gas typeor composition, ranging from 60 μm to about 400 μm.

With the pore diameter and the membrane wall thickness of the membranestructure to be treated according to the present invention, plasmaignition will normally take place only on the outer side of the membraneor, in certain circumstances, e.g. in the presence of helium, on thelumen side, when the contact between the excited gas molecules and thewall is minimised due to an axial laminar helium gas flow andapplication of a wobbling frequency with high harmonic overtoneadditives. When there is no pressure gradient between the outer side ofthe membrane and the lumen side, the activated particles can enter thepore structure only by diffusion from the plasma zone (may be outside inmost cases, or from the lumen side in special cases). The diffusingactivated particles will collide with gas molecules and with the wallsof the pores on their way from the plasma zone into the pore structureand dissipate their energy. The amount of gas molecule or wall contactswhich is necessary for losing the activating energy could previouslyonly be determined empirically. In consequence, there will be adecreasing chemical modification density of the pore walls from theplasma zone area into the membrane structure. The chemical plasmamodification density distribution can be influenced by the poregeometry, the plasma intensity, the pressure, the gas composition, thepressure difference over the membrane structure, and the power spectrumof electric frequency input.

The regioselective introduction of the functional groups can be achievedin four different ways for microporous hollow fibre membrane substrates,comprising four different embodiments of the process according to thepresent invention, as appears from FIGS. 2 a-2 d, i.e. 2 a) outside lowpressure treatment (diffusion control) 2 b) outside high pressuretreatment (laminar or convective control) 2 c) inside low pressuretreatment (laminar or convective control) 2 d) inside high pressuretreatment (diffusion control).

The processes shown in FIGS. 2 a and 2 b represent a first main mode andprovide a regioselective functionalisation of the outer surface and thepore surface; the processes shown in FIGS. 2 c and 2 d represent asecond main mode and provide a regioselective functionalisation of theinner surface and the pore surface. Thus, these four differentembodiments (or two main modes) are intended for different uses, i.e.depending on if the lumen surface is intended to be on the blood side orthe filtrate side of the microporous hollow fibre membrane.

As appears from FIG. 2 a, showing one embodiment of outside low pressuretreatment, diffusion controlled outside plasma treatment under lowpressure (0.1-10 mbar), preferably about 1.6 mbar (0.3 mbar modifyinggas)) is performed by adding the gas mixture to the outside of themicroporous hollow fibre membrane substrate. A fibre module of a hollowfibre membrane substrate is placed between two electrodes, preferablyring electrodes, around a polycarbonate housing. Openings in the housingallow a gas flow along the outer surface of the membrane substrate.After appropriate evacuation the gas mixture is introduced and ignitionis performed creating a gas plasma mixture. The gas plasma mixturepenetrates the membrane substrate structure by diffusion, i.e. thedriving force from mass transfer equals the concentration gradient. Theprocess preferably involves one to ten cycles of plasma ignition at13.56 MHz during 1 to 10 sec under the gas mixture atmosphere, followedby a plasma-off period of 2-3 minutes. During the flow of the gas plasmamixture functional groups, e.g. amino groups, are attached to the outersurfaces and the pore surfaces of the hollow fibre membrane substrate.Finally, the fibre modules are evacuated for 1-60 min, normally about 15min, to remove non-adsorbed modifying gas present in the gas mixture.The inlet and outlet openings for the gas mixture are preferably locatedat the opposite ends of the housing. This embodiment gives highlysatisfactory results as to regioselective affinity for a hollow fibremembrane for whole blood treatment and is therefore the most preferredembodiment of the present invention.

As appears from FIG. 2 b outside plasma treatment under high pressure(50 mbar-1.1 bar) is performed in the same way as for the low pressuretreatment except for the fact that the gas plasma mixture penetrates themembrane substrate structure by convection or laminar flow.

As appears from FIG. 2 c convection or laminar flow controlled insideplasma treatment under low pressure (0.01-50 mbar) is performed byadding the gas mixture into both ends of the fibre bundle, wherein thegas mixture penetrates the pores from the lumen side to the outer sideof the hollow fibres, i.e. to the polycarbonate housing space, and thenexits the housing space through the gas mixture exits arrangedperpendicular or substantially perpendicular to the fibre bundledirection. Further, the electrodes are preferably arranged in such a waythat the gas mixture exits are arranged between the electrodes.

As appears from FIG. 2 d diffusion controlled inside plasma treatmentunder high pressure (50 mbar-1.1 bar) is performed by adding gas mixtureat one end of the fibre bundle, wherein the gas mixture exits shown inFIG. 2 c are closed and the concentric polycarbonate housing or casingsurrounding the fibre bundle is filled with a blocking fluid, e.gpolyethylene glycole, thereby allowing the gas mixture to more or lessfill the pores but preventing it from passing out from the pores to theouter surface. Instead, the gas mixture exits the fibre bundle at theopposite end.

In the process for preparation of a microporous hollow fibre membraneaccording to the present invention, the ignition frequency during theplasma ignition is 1 kHz-13.56 MHz or multiples of 13.56 mHz ormicrowave frequency, the power is 0.5-20 W, the voltage of theelectrodes is 50-500 volts, the pressure is 0.01-10 mbar, the flow rateis 0.1-200 sccm/min, and the gas plasma mixture flow period is up to 20min.

The plasma treatment experiments and the analyses described below werecarried out, if not otherwise stated, for a microporous hollow fibremembrane having regioselective affinity produced according to the mostpreferred embodiment according to the present invention, i.e. whereinDACH/helium as gas mixture was added to the membrane substrate duringthe plasma treatment.

For microporous flat sheet membranes the regioselective introduction ofthe functional groups is achieved as follows.

FIG. 3 shows the preparation of a microporous flat sheet membrane havingregioselective affinity for undesired compounds in blood or otherbiologically active fluids by use of plasma ignition. The flat sheetmembrane substrate is enclosed in a housing or casing, having a firstand a second compartment separated from each other by the flat sheetmembrane substrate. During the plasma ignition treatment the gas mixtureis initially introduced in the first compartment, also comprising aplasma chamber with two electrodes connected to a power supply. Afterthe plasma ignition of the gas mixture the gas plasma-mixture obtainedflows against and passes the flat sheet membrane substrateperpendicularly arranged in relation to the gas plasma mixture flow. Theflat sheet membrane substrate surface facing the first compartment, i.e.on the intended filtrate side of said membrane substrate, and the poresurfaces are regioselectively provided with functional groups. Nofunctional groups are bound to the flat sheet membrane substrate surfacefacing the second compartment, i.e. on the intended blood side of themembrane. Excess gas continues to flow through the second compartmentand is then evacuated therefrom. A vacuum pump connected to the secondcompartment provides the flow through the whole arrangement. Appropriateligands are then bound to the functional groups in a conventional way.

In the process for preparation of a microporous flat membrane accordingto the present invention, the ignition frequency during the plasmaignition is 1 kHz-13.56 MHz or multiples of 13.56 mHz or microwave, thepower is 1-20 W, preferably about 5 W, the voltage of the electrodes is50-300 volts, the pressure is 0.1-5 mbar, preferably about 0.3 mbar, theflow rate is 1-100 sccm/min, preferably 10 sccm/min, and the gas plasmamixture flow period is up to 30 min, preferably about 5 min. Theparameters during this plasma ignition treatment are further describedin Example 3.

It is to be understood that the housings or casings, inlets, outlets,electrodes etc in the devices shown in FIGS. 2 a-2 d and 3 may bealtered as to size, mutual arrangement, type and geometry, still givingthe beneficial effects desired for the present invention.

An electron spectroscopy for chemical analysis (ESCA) was performed witha view to quantitatively evaluating the amino group distributionresulting from the plasma treatment.

First a sample of a microporous affinity membrane with regioselectiveaffinity is illuminated with Al k-alpha rays (1486.6 eV), and the energyof emitted electrons is measured. Fluorine is used only as a marker forthe functionality arranged at the membrane surfaces, which itself doesnot contain any fluorine. Instead, the membrane surface functionalitiesare derivatised with a fluorine containing compound, e.g.pentafluorobenzaldehyde, with a view to quantifying the functionalgroups bound to the membrane surfaces.

The derivatisation procedure is preferred as follows: 300 μl stocksolution of 0.1 M PFBA in pentane is added to 15 ml pentane. Afteraddition of the test material the solution is brought to react during 2hours at 39° C. in a water bath and under reflux. This is followed bywashing during the night in pentane in a Soxhlet device at 43° C. (onecycle: 20 min).

In the table below the distribution of atoms in the functional groups onthe outer (shell) and inner (lumen) surface of microporous hollow fibremembranes is shown. It appears that, due to the preferred embodiment ofthe process according to the present invention, the presence of primaryamino groups on the inner surface is zero (no fluorine-signal!). The 1.4atom % nitrogen is due to the PVP content of the membrane. TABLE Atomdistribution (ESCA) of plasma-modified PES/PVP membranes afterderivatisation with pentafluorobenzaldehyde Distribution of elements [%]Surface C O S N F Shell 73.0 10.2 0.7 8.3 7.8 Lumen 74.5 21.7 2.4 1.4 —

Further, an ESCA analysis was performed with a PP membrane treated withdifferent plasma treatment modes. The table below shows the atomdistribution of plasma treated (DACH) PP membrane substrates on inner(lumen) and outer (shell) surfaces of hollow fibre membranes.

Atom Distribution (ESCA) of Plasma Treated (DACH) PP Membranes

N may here be used as marker as PP does not contain N. Distribution ofelements [%] Treatment mode Surface C O N Gas stream outside, shell 90.06.9 3.1 parallel to fibres lumen 94.8 5.2 — (diffusion controlled) Gasstream through shell 84.7 7.2 8.1 the membrane wall lumen 94.2 4.5 1.3of hollow fibre (convection controlled)

This indicates an approximate 5-fold surplus of amino groups on theouter surface relative to the inner surface for a convection controlledprocess and the absence of amino groups on the inner surface for adiffusion controlled process.

Moreover, the table below shows the concentration of introduced activeamino groups depending on the treatment mode used to introduce them fora PES/PVP hollow fibre membrane. NH₂ concentration Treatment mode[mmol/g] Outside plasma, low pressure (FIG. 2a) 0.08-0.09 Outsideplasma, high pressure (FIG. 2b) 0.03 Inside plasma, low pressure (FIG.2c) 0.02 Inside plasma, high pressure + blocking 0.06 fluid (FIG. 2d)

The highest concentrations are achieved with the outside plasma/lowpressure mode treatment (see FIG. 2A). These concentrations come closethe ones required for the monomolecular immobilisation of peptides ofseveral 1000 Da.

Thus, regioselective modification of membrane substrates with a higherselectivity for the outer surface can be achieved. This makes themembranes interesting for arranging ligands selectively on theirsurfaces. As stated above the regioselectively arranged ligands enable aselective removal of toxins or other target compounds by adsorptionduring therapeutic purification of blood or other biologically activefluids, while the interaction of constituents in blood or such fluidswith the ligands or adsorbed toxins is avoided.

Examples of compounds of interest to remove from blood or otherbiologically active fluids are e.g. endotoxins and inflammatorymediators in septic patients, pathogenic antibodies in several immunediseases, low-density lipoproteins in patients with coronary heartdisease and drug resistant hypercholesterolemia, and fibrinogen used forthe treatment of microcirculatory disorders.

The present invention also relates to use of the microporous affinitymembrane produced according to the present invention and havingregioselective affinity in therapeutic apheresis, for diagnosticapplications when enrichment of trace materials is necessary (e.g.pesticides in food or water, metabolites and drugs in plasma, urine, andsaliva), and for drug development applications. Common for thesedifferent applications is that blood constituents are not activatedduring the use of the microporous affinity membrane.

In the following examples of the process according to the presentinvention, functionalisation, i.e. providing regioselective affinity,with amino groups for a single hollow fibre, a fibre bundle modificationand a flat sheet membrane substrate, respectively, is shown for PES-PVPmicrofiltration membranes.

Example 1 Single Hollow Fibre Modification

The plasma treatment mode shown in FIG. 2 a) was used. The fibre lengthwas 15 cm and the tube diameter 1.2 cm. The system was evacuated at apressure below 0.01 mbar during 15 min. DACH was added at a flow rate of0.5 sccm/min; applicable range: 0.1-200 sccm/min) at a pressure of 0.3mbar (applicable range: 0.1-10 mbar). The plasma ignition was performedat 13.56 MHz (1 kHz to 13.56 MHz) and multiples of 13.56 MHz andmicrowave at 15 W (applicable range: 0.5-200 W) during 1 sec (applicablerange: 0.1 sec-10 min). After the plasma treatment step the system wasflushed with H₂ at 10 mbar during 5 min, followed by venting with N₂ tominimize oxidation of the membrane.

Example 2 Fibre Bundle Modification (50 Hollow Fibres)

The steps in Example 1 were repeated with the exceptions that 2 sccm/min(applicable range: 1-100 sccm/min) helium was added as carrier gastogether with the DACH, that the total pressure was 1.2 mbar (applicablerange: 0.1-10 mbar), that the effect at the plasma ignition step was 2 W(applicable range: 1-20 W) and that the plasma time was 15 min(applicable range: 10 sec-30 min). This parameter set results in properamino functionalisation of outer surfaces and inner pore surfaces of all50 hollow fibres.

Example 3 Modification of a Microporous Flat Sheet Membrane Substrate

The plasma treatment mode according to FIG. 3 was used. The system wasevacuated at a pressure below 0.01 mbar. H₂ was added at a flow rate of10 sccm/min together with DACH at a total pressure of 0.3 mbar. Theplasma ignition was performed at 13.56 MHz and an effect of 5 W and theplasma time was 5 min.

The result obtained is a flat sheet membrane regioselectivelyfunctionalised with amino groups on the surface on the filtrate side andon the pore surfaces, but not on the surface on the blood side.

1. A process for production of a microporous affinity membrane havingregioselective affinity for compounds in blood or other biologicallyactive fluids to be removed during purification of blood or saidbiologically active fluids, comprising subjecting a microporous affinitymembrane substrate having a blood side and a filtrate side to one ormore cycles of plasma ignition in the presence of a gas mixturecomprising at least one modifying gas, wherein the modifying gascomprises at least one functional group, and wherein the at least onefunctional group is regioselectively bound to pore surfaces of themicroporous affinity membrane substrate.
 2. The process according toclaim 1, wherein the microporous affinity membrane substrate is amicroporous hollow fibre membrane substrate.
 3. The process according toclaim 1, wherein the microporous affinity membrane substrate is amicroporous flat sheet membrane substrate.
 4. The process according toclaim 1, wherein ligands having affinity for the compounds in blood orother biologically active fluids are bound to the at least onefunctional group.
 5. The process according to claim 1, wherein the atleast one functional group is regioselectively bound to surfaces on thefiltrate side of the microporous affinity membrane substrate.
 6. Theprocess according to claim 4, wherein the ligands are proteins,peptides, amino acids, carboxylic acids, nucleotides, oligonucleotides,antigens, antibodies, or mixtures of two or more thereof.
 7. The processaccording to claim 1, wherein the at least one functional groupcomprises an amino, aldehyde, ester, epoxy, hydroxy, or sulfonic acid.8. The process according to claim 7, wherein the at least one modifyinggas is diaminocyclohexane (DACH) or diethylenetriamine (DETA).
 9. Theprocess according to claim 1, wherein the gas mixture also comprises atleast one carrier gas.
 10. The process according to claim 9, wherein theat least one carrier gas is chemically inert during the process.
 11. Theprocess according to claim 1, wherein the plasma ignition results in agas plasma mixture with a flow rate of 0.1-200 sccm/min.
 12. The processaccording to claim 9, wherein the proportion between the at least onemodifying gas and the at least one carrier gas is 1:100 to 1:1.
 13. Theprocess according to claim 1, wherein up to 10 cycles of plasmaignitions are performed.
 14. The process according to claim 2, whereinthe microporous hollow fibre membrane substrate is enclosed in a housingor a casing throughout the process.
 15. The process according to claim2, wherein the plasma ignition results in a gas plasma mixture flowingaxially along the outer or inner surface of the microporous hollow fibremembrane substrate.
 16. The process according to claim 2, wherein themicroporous hollow fibre membrane substrate is made up of a mixture ofpolyethylenesulfide and polyvinylpyrrolidone having an inner diameter of200-1000 μm, a wall thickness of 20-200 μm, a pore diameter of 0.1-0.8μm, and is assembled in modules each having 1 hollow fibre or assembledin bundles or modules of more than 1000 fibres.
 17. The processaccording to claim 2, wherein the ignition frequency during the plasmaignition is 1 kHz-13.56 MHz or multiples of 13.56 mHz or microwavefrequency, the power is 0.5-20 W, the voltage of the electrodes is50-500 volts, the pressure is 0.01-10 mbar, the flow rate is 0.1-200sccm/min, and the gas plasma mixture flow period is up to 20 min. 18.The process according to claim 14, wherein the gas mixture is added tothe housing or casing space surrounding the outer surface of themicroporous hollow fibre membrane substrate in a diffusion controlledway at a pressure of 0.01-50 mbar.
 19. The process according to claim14, wherein the gas mixture is added to the housing or casing spacesurrounding the outer surface of the microporous hollow fibre membranesubstrate in a laminar flow or convection controlled way at a pressureof 50 mbar-1.1 bar.
 20. The process according to claim 2, wherein thegas mixture is added to the lumen of the microporous hollow fibremembrane substrate in a laminar or convection controlled way at apressure of 0.01-50 mbar.
 21. The process according to claim 14, whereinthe gas mixture is added to the lumen of the microporous hollow fibremembrane substrate in a diffusion controlled way at a pressure of 50mbar-1.1 bar, and wherein the housing space surrounding the outersurface of the microporous hollow fibre membrane substrate is filledwith a blocking fluid.
 22. The process according to claim 3, wherein themicroporous flat sheet membrane substrate throughout the process isenclosed in a housing or casing having a first and a second compartmentseparated from each other by said membrane substrate, wherein thesurface on the filtrate side of said membrane substrate is facing thefirst compartment and the surface of the blood side is facing the secondcompartment, and wherein the gas mixture is added to said firstcompartment and the functional groups during the plasma ignition in thepresence of the gas mixture are bound to pore surfaces and the surfaceon the filtrate side of the microporous flat sheet membrane substrate.23. The process according to claim 22, wherein the plasma ignitionresults in a gas plasma mixture with a flow rate of 1-100 sccm/min. 24.The process according to claim 3, wherein the microporous flat sheetmembrane substrate is made up of a mixture of polyethersulfone andpolyvinylpyrrolidone having a wall thickness of 20-200 μm.
 25. Theprocess according to claim 3, wherein the ignition frequency during theplasma ignition is 1 kHz-13.56 MHz or multiples of 13.56 mHz ormicrowave, the power is 1-20 W, the voltage of the electrodes is 50-300volts, the pressure is 0.1-5 mbar, the flow rate is 1-100 sccm/min, andthe gas plasma mixture flow period is up to 30 min.
 26. The processaccording to claim 22, wherein excessive gas is evacuated from thehousing or casing spaces after the plasma ignition.
 27. A microporousaffinity membrane produced according to claim 1, wherein saidmicroporous affinity membrane comprises at least one functional group,bound only to pore surfaces of the microporous affinity membrane. 28.The microporous affinity membrane according to claim 27, wherein the atleast one functional group comprises an amino group.
 29. The microporousaffinity membrane according to claim 27, wherein the at least onefunctional group is bound to the filtrate side.
 30. The microporousaffinity membrane according to claim 27, wherein ligands havingspecificity for the components in blood or other biologically activefluids to be removed are bound to the functional groups.
 31. Themicroporous affinity membrane according to claim 27, wherein themicroporous affinity membrane is a microporous hollow fibre membrane ora microporous flat sheet membrane.
 32. A microporous affinity membraneaccording to claim 30, wherein the ligands are proteins, peptides, aminoacids, carboxylic acids, nucleotides, oligonucleotides, antigens,antibodies, or mixtures of two or more thereof.
 33. An adsorption devicecomprising the microporous affinity membrane according to claim 27.34-37. (canceled)
 38. The process according to claim 1, wherein the atleast one functional group comprises an amino group.
 39. The processaccording to claim 9, wherein the at least one carrier gas compriseshelium, nitrogen, hydrogen, argon, or a mixture of two or more thereof.40. The process according to claim 9, wherein the proportion between theat least one modifying gas and the at least one carrier gas is 1:4. 41.The process according to claim 14, wherein the housing or casing isconcentric.
 42. The process according to claim 2, wherein themicroporous hollow fibre membrane substrate is made up of a mixture ofpolyethylenesulfide and polyvinylpyrrolidone having an inner diameter ofabout 330 μm, a wall thickness of about 110 μm, a pore diameter of about0.4 μm, and is assembled in modules each having 1 hollow fibre orassembled in bundles or modules of more than 1000 fibres.
 43. Theprocess according to claim 2, wherein the microporous hollow fibremembrane substrate is assembled in bundles or modules of up to 1000fibres.
 44. The process according to claim 23, wherein the flow rate isabout 10 sccm/min.
 45. The process according to claim 24, wherein themicroporous flat sheet membrane substrate has a wall thickness of about110 μm, and a pore diameter of about 0.4 μm.
 46. The process accordingto claim 25, wherein the power is about 5 W, the pressure is about 0.3mbar, the flow rate is 10 sccm/min, and the gas plasma mixture flowperiod is about 5 min.
 47. A method of therapeutic apheresis, comprisingtreating blood or other biologically active fluids with the microporousaffinity membrane according to claim
 27. 48. The method of claim 47,wherein blood constituents are not activated.
 49. A method of diagnosingthe presence of a compound in a material comprising blood or otherbiologically active fluids, food, or water, comprising detecting thecompound in the material with the microporous affinity membraneaccording to claim
 27. 50. The method of claim 49, wherein, whendetecting the compound in blood or other biologically active fluids,blood constituents are not activated.
 51. A method of drug development,comprising detecting a potential drug compound in blood or otherbiologically active fluids with the microporous affinity membraneaccording to claim
 27. 52. The method of claim 51, wherein bloodconstituents are not activated.
 53. A method of purifying blood or otherbiologically active fluids, comprising comprising treating the blood orother biologically active fluids with the microporous affinity membraneaccording to claim
 27. 54. The method of claim 53, wherein bloodconstituents are not activated.